The present invention concerns the use of pirenoxine for the protection of the corneal tissues in photokeratectomy interventions. More particularly, the invention concerns the use of pirenoxine and salts thereof as agents able to inhibit within the cornea the oxidative phenomena determined by reactive oxygen species (or ROS, reactive oxygen species) which are produced in the tissues following the laser irradiation.
As known, the ophthalmic surgery, and particularly the refractive one, which aims to modify the eye refractive power in order to correct not negligible visual defects makes use of various, less or more consolidated or in evolution techniques, some examples of which are radial keratectomy, epikeratofachia and keratomileusis. In addition to these, also in the ophthalmology field the use of laser, particularly solid state laser, (like neodymium:yttrium-aluminiumrnarnet laser, known as Nd:YAG), and, above all, excimer laser, is remarkably increased.
Excimer laser is a pulse laser which, due to the decay of excited noble gas dimers (excimers obtained from gas mixtures of halogen and noble gases), are able to emit large amounts of energy in form of radiation within the range of far ultraviolet (UV-C), in the form of pulse trains having predetermined duration, frequency and fluence. Any photon emitted during the irradiation has enough energy to break the intramolecular bonds of the exposed material, in such a way that the irradiated molecules are xe2x80x9cbrokenxe2x80x9d in small volatile fragments which are expulsed at supersonic speed embodying a process known as xe2x80x9cphotodecompositionxe2x80x9d.
In the applications using the excimer laser in corneal surgery interventions usually an argon-fluorine laser, emitting radiation with a wavelength of 193 nm, which is suitable to carry out highly precise interventions with an optimal control on the penetration depth and a minimal thermal or mechanical damage effect on adjacent to exposed tissues, is usually employed. Contrary to other lasers used in clinical field, the excimer laser does not emit energy concentrated in a focal point but it has a radius with a large cross section which, going through suitable slits, is directed to strike large surface cornea zones with an accurate control of the shape and sizes of the exposed zones. The emitted energy is almost totally adsorbed by a surface layer within a thickness of few microns and results, by means of evaporation, in ablation at every pulse of cornea layers little more thicker than molecular, with a reproducibility not attainable by other techniques.
The excimer laser is widely used for corneal refractive re-modelling in techniques known as photorefractive keratectomy or PRK and LASIK (laser intrastromal keratomileusis), for the correction of various ametropias among which the most diffused is myopia. As known, the latter is a defect determined by a cornea curvature higher than required by the length of ocular bulb, so that light rays from outside are refracted in a such way that, before to reach the retina, they converge in a focal point. In this circumstance the use of excimer laser provides that layers of corneal tissue, the thickness of which is increasing toward the centre, be ablated reducing therefore the curvature of the cornea. When the technique is used for the correction of hypermetropia, wherein, on the contrary, the modification to be obtained is an increase of the cornea curvature, the amount of ablated tissue within the periphery of the exposed zone is more important than in the centre. Finally for the correction of the astigmatism which, as known, is an ametropia caused by curvature difference in various meridians of the ocular surface, the depth of the ablation can be asymmetric, depending on the meridian to be xe2x80x9cflattedxe2x80x9d.
More recently the use of the excimer laser has been suggested for the therapeutic removal of surface corneal tissues, for the treatment of various corneal irregularities and opacities: like of dystrophic, degenerative, cicatricial or infective type. Such an operation, called phototherapeutic keratectomy or PTK, has been used, for example, for the treatment of recurrent corneal erosions, post-operation kerattis, corneal dystrophies as Reis-Buckler dystrophies, corneal opacities or cicatrices caused by Herpes simplex, surface irregularities following surgical interventions, for example as outcomes from keratoplasty or refractive corneal interventions. Contrary to refractive photokeratectomy PTK aims to eliminate irregularities on the corneal surface in order to flat the profile thereof and therefore involves the ablation of tissue layers with different thickness in the various zones of treated corneal surface.
Although the above described photokeratectomy interventions appear to be an alternative less traumatic than surgical ophthalmic techniques, the restorative process after the photoablation is not without drawbacks which are less or more transitory and boring or dis-enabling for the patient, among which, for example, there are corneal cicatricial problems, generation of under-epithelial opacities called xe2x80x9chazexe2x80x9d, which determine a reduction of visual efficiency resulting from xe2x80x9clight scatteringxe2x80x9d phenomenon (light diffusion) and, in some circumstances, a reduction of refractive values as result of operation. It appears to be not debatable by those skilled in the field that at least partially such effects result from the formation of free radicals and, generally, reactive oxygen species, which was detected as side effect of UV irradiation and temperature increase occurring in the involved tissues.
As known the term xe2x80x9creactive oxygen species (or substances)xe2x80x9d, or ROS, presently collectively means the free radicals and not radical chemical species which currently take part into oxidative biological processes and whose excess with respect to the natural equilibrium conditions is considered to be the base of an ever increasing number of degenerative and pathological phenomena. Specifically the term ROS comprises superoxide anionic radical O2., hydroxyl radical OHxe2x88x92, singlet oxygen 1O2 and the hydrogen peroxide, H2O2, as well as alkoxide RO. and peroxide ROO. radicals which are generated from organic molecules during the oxidative processes. The activity of these species exerts, within the organism, on various cellular components, among which there are a large number of structural proteins and enzymes, DNA, RNA and, above all, the membrane lipids.
In fact the lipid peroxidation is the most known mechanism by which ROS exert their degenerative activity on the cellular structures damaging polyunsaturated fatty acids (PUFA) contained in the cytoplasmic membranes, often as phospholipid esters. In the initial step of this process the action of a free radical abstracts an hydrogen atom H. from the lipid chain, forming a free radical R* which undergoes a molecular rearrangement of the double bonds resulting in a conjugated diene radical. The latter rapidly reacts with molecular oxygen forming thus a lipid peroxide radical ROO., which, being a so strong oxidant to attack another PUFA, starts the propagation step of the reaction. In such a way a lipid hydroperoxide radical, ROOH; and, correspondingly, another lipid peroxide radical ROO., are formed. Therefore the above described main branch of the reaction occurs by means of radical chain attacks to the membrane lipids which are thus transformed step by step in the corresponding hydroperoxides till to the chain termination by means of a free radical.
Various agents naturally occurring in the cellular tissues can perform the above described action, practically functioning as scavengers or antoxidants. Among these the most known are C (ascorbic acid) and E (alpha tocopherol) vitamins, antioxidant enzymes as superoxide dismutase (SOD), catalase, gluthatione peroxidase and various low molecular weight compounds, among which gluthatione (GSH), tyrosine, uric acid. The natural protection from oxidative stresses performed by these substances, however, can not be enough strong to antagonize the degradation effect of ROS, in which circumstance the lipid peroxidation can result in an irreversible damage to the cellular membranes.
It has been also demonstrated that the oxidized forms of transition metal ions, as Fe3+ and Cu2+, in the presence of H2O2, can accelerate the oxidative mechanism by a non enzymatic reaction known as Fenton reaction. In the presence of a reducing agent, as ascorbate, part of the oxidized ions is reduced to the lower oxidation state (for example Fe2+) and the reaction, whose rate depends on the Fe3+:Fe2+ ratio, proceeds, resulting in the conversion of hydrogen peroxide in hydroxyl ion, OHxe2x88x92, plus an hydroxyl radical, OH. The latter represents the most reactive ROS.
Although it is difficult to detect ROS due their reactivity and therefore their short life times, the formation of free radicals in tissues subjected to photoablation using excimer laser has been widely demonstrated. For example the presence of free radicals in bovine corneas exposed to irradiation using ArF laser has been revealed by EPR spectroscopy (electron paramagnetic resonance) (R. J. Landry et al., Laser and Light in Ophthalmol., 6: 87-90, 1994), while measurements of temperature increase at level of the corneal endothelium and analytical determinations of the reduction of the SOD activity at level of the aqueous humour confirmed the formation of ROS in the cornea of PRK treated rabbits (K. Bigihan et al., Jpn. J. Ophthalmol., 40, 154-157, 1996). The lipid peroxidation has been detected, again in the rabbit cornea, following PTK treatment performed using excimer laser, both by histochemical test and the analytical detection of the presence of degradation products in corneal lipid extracts, particularly conjugated dienes and ketodienes (S. Hayashi et al., British J. Ophthalmol. 81, 141-144, 1997).
Further it has been pointed out by EPR spectroscopy the generation of free radicals also when corneal tissues are irradiated using solid state Nd:Yag laser, at a wavelength of 213 nm rather than 193 nm, which wavelength is typical of the argon-fluorine excimer laser. However in this case in addition to an oxidative damage comparable to that obtained using the excimer laser, it has been also detected a more remarkable cytotoxic effect, somehow dependent on the higher wavelength of the radiation (E. Edigeretal., Lasers Surg. Med., 21:88-93, 1997).
In addition to the effect of the UV radiation on the primary production of ROS, it has been also observed that the chemiotaxis activity of thus formed lipid hydroperoxides withdraws in situ polymorphonucleated cells and macrophages which in turn, by producing further ROS, enhance the damaging action of the radiation inducing a set of cytotoxic effects (H. Goto et al., Curr. Eye Res., 10:1009-1014, 1991).
Although the above reported literature demonstrates the formation of free radicals and reactive oxygen species in the photoablation treatment and relates this phenomenon to other possible post-operation complications it is not considered to be particularly important the protection of the corneal tissues by administration of exogenous agents having ROS antagonizing activity both before and after the operation. In effect the currently used pharmacological therapy for the photokeractectomy treatments is consisting of the topic ocular application, after the operation, of antibiotics, with the clear purpose to maintain in aseptic conditions the ocular surface during the cicatrization process, and anti-inflammatory drugs (steroidal or, according to the most recent trends, non steroidal) in order to act against the post-operation phlogosis conditions.
Therefore the object of the present invention is to provide the corneal tissues involved in UV irradiation, both before and soon after the treatment, with an agent suitable to perform a protective activity against the cellular damage triggered by the reactive oxygen species and to scavenge the action thereof. Particularly the suggested agent must be effective to oppose the lipid peroxidation in the corneal cellular tissues.
Within the studies about the effects of ROS and the inhibition of lipid peroxidation by various exogenous molecules having scavenging or antioxidant activity it has been found out that pirenoxine, an active principle already known and used therapeutically on another ocular district, the crystalline lens, shows a remarkable activity for the inhibition of lipid peroxidation in the comeal tissues and it is therefore able to perform a protective action against the cellular modifications resulting form laser irradiation.
Pirenoxine or 1-hydroxy-5-oxo-5H-pyrido-[3,2a]-phenoxazin-3-carboxylic acid (also called pirfenossone) is a known compound having the following formula: 
used in ophthalmology, usually in the form of sodium salt thereof, for the treatment of the cataract. The latter is an abnormal progressive condition of the eye crystalline lens characterized by an increasing loss of transparency. As known, the cataracts more often result from degenerative modifications, often occurring after 50 year age, while more rarely they can result from traumas or poison exposure. Initially the vision is hazy, then the brilliant lights dazzle diffusely and distortion and double vision can develop. At the end, if the cataract is not treated, anopia occurs. In addition to the surgical treatment, which becomes necessary for more advanced degenerative states and involves the ablation of the crystalline lens (with or without surgical implantation of an intraocular lens) the cataracts can be treated by the ophthalmic topic administration of pirenoxine in the form of collyrium.
It has been postulated that the ability of pirenoxine to inhibit the formation of lenticular opacities results from at least three different action mechanisms: (a) inhibition of the oxidation activity of the quinone molecules on the lenticular proteins, by binding their xe2x80x94SH groups; (b) activation and normalization of the cation pumping activity performed by the capsule of the crystalline lens; (c) inhibition of the sorbitol synthesis and reduction of the osmotic damage resulting from the storage of this substance (S. Iwata, J. Pharmac. Soc. Jap., 1964; 844: 435-440; F. Ikemoto et al., in: Proc. 50th Congr. Pharmacol. Soc. Jap., Kanto Region, 1974: I. Korte et al., Ophthalmic Res., 1979; 11: 123-125).
Within the most recent studies about the biological activity of pirenoxine it has been also found out, and it is the object of the european patent application No. EP 0885612, assigned to the present Applicant, that this molecule, in addition to the activity in the treatment of cataract, is has anti-inflammatory properties too. These properties, which have been verified on animal models, embody through an action mechanism not elucidated in the mentioned patent application, although in the above mentioned patent description it is postulated an inhibiting activity of the oxidative catabolism of arachidonic acid, which results in the production of prostaglandins.
According to the present invention it has been found out, as already reported, that pirenoxine can be advantageously used for the protection of the corneal tissues during excimer laser treatments because it is active in inhibiting the lipid peroxidation in the corneal cellular tissues.
It is therefore an object of the present invention the use of 1-hydroxy-5-oxo-5H-pyrido-[3,2a]-phenoxazin-3-carboxylic acid (pirenoxine) or a pharmaceutically acceptable salt thereof for the production of a topic ophthalmic drug suitable for the protection of the corneal tissues in photokeratectomy interventions. As already pointed out the suggested drug is designed as inhibitor of the ROS activity (reactive oxygen species) at level of comeal tissues and, particularly as inhibitor of the lipid peroxidation at level of said tissues.
Use of pirenoxine as pre- and post-operation protective agent finds application in any photokeratectomy treatment, being further presumable a wider use in those treatments which presently are more diffused, i.e. corneal photoablation by means excimer laser, both refractive and therapeutic and, in the first case by means of both PRK and LASIK technique.
The ophthalmic preparations of the present invention preferably contain the active principle, i.e. pirenoxine or a pharmaceutically acceptable salt thereof, in amount from 0,0001% to 0,01 weight %, expressed as free acid. More conveniently said medicaments contain from 0,001% to 0,005 weight % of pirenoxine, expressed as free acid, the optimum concentration being the same as that presently used for the therapy of the cataract, i.e. 0,005 weight %. Most conveniently said pirenoxine is in the form of the sodium salt. When used in the form of collyrium containing 0,005 weight % of the active principle, the preparation according to the invention can be administered, in order to obtain the desired effect of ROS inhibition, at a dosage of one-two drops twice or three times a day, preferably two drops three times a day, beginning at least one or two days before the operation and continuing, after the operation, over at least one or two days. Generally the dosage and posology can be widely variable without impairing the whole protective effect against ROSs exerted by the product.
The ophthalmic topic drug containing pirenoxine or a salt thereof can be, generally, in the same forms as prepared or proposable for the use of the same active principle for the therapy of the cataract or ophthalmic inflammation, as described in the above mentioned european patent publication EP-A-0885612. Particularly, the product can be in the form of aqueous solution or suspension for collyrium or in the form of emulsion, ointment, gel or cream. Preferably the product is administered as aqueous ophthalmic solution. Because of the instability of the active principle, pirenoxine is normally formulated, in the already used medicaments for the treatment of the cataract, as a two component preparation wherein a first component comprises freeze-dried pirenoxine and the second component comprises an eye acceptable aqueous carrier or diluent. The two components are reconstituted before the use and the thus obtained solution can be generally stored at ambient temperature for about two weeks without degradation.
Generally the compositions containing pirenoxine or a salt thereof according to the invention can be formulated according to the known art, for example according to the teachings suggested by xe2x80x9cRemington""s Pharmaceutical Sciences Handbookxe2x80x9d, Hack Publ. Co., U.S.A. Usually one or more agents for the regulation of tonicity should be added whereby the solution has a suitable osmolarity value. Any one of the products usually used in the art can be used, as, for example, sodium chloride, potassium chloride, mannitol, dextrose, boric acid, propylene glycol. The preparation can also comprise acids or bases as agents for the regulation of pH and/or buffers, as, for example, monosodium phosphatexe2x80x94disodium phosphate, sodium boratexe2x80x94boric acid or sodium succinatexe2x80x94succinic acid systems. For a good tolerability in the eye the pH should be between 4,5 and 8,5. Furthermore the composition should also comprise preservatives and antimicrobial agents, as benzalkonium chloride, sodium merthiolate or thimerosal, mehyl-, ethyl- and propyl-paraben, chlorobutanol, as well as chelating or sequestering agents as edetates or EDTA. If the product is packaged in unit dose containers the presence of preservatives can be avoided but, when multiple dose containers are used, for example vials for collyrium containing from 5 to 15 ml, the presence of the preservatives is necessary.
In addition the ophthalmic preparation can comprise further optional ingredients, as thickening agents, anti-oxidants, stabilizers, surface active agents, ecc. Only for exemplary purpose the composition of an already commercially available product designed for the treatment of the cataract is described below. The formulation can be suitable also for the use of the product as cornea protective agent against free radicals and ROS.